PROJECT SUMMARY Substance abuse, including the abuse of psychostimulants and opiates, represents a major public health concern that exacts tremendous financial and societal costs. Abuse of these drugs remain recalcitrant conditions with limited FDA-approved treatments. Elucidation of the differential neural molecular mechanisms underlying psychostimulant versus opiate abuse is necessary to the development of novel, targeted therapies. There is a substantial body of evidence indicating that epigenetic and transcriptional mechanisms contribute to the pathogenesis of both psychostimulant and opiate abuse and dependence. Changes in the expression levels and the genomic localization of transcription factors have been identified in drug-exposed animal models. For example, repeated exposure to opiates or to psychostimulants elevate cyclic AMP-responsive element binding protein (CREB)-mediated transcription in the nucleus accumbens (NAc), a major brain reward region. Over-expression of CREB decreases the rewarding effects of psychostimulants and opiates, suggesting that CREB activity in this region can regulate the addictive properties of these drugs. However, manipulations of this kind cause epigenetic and transcriptional changes at hundreds of loci, limiting mechanistic insight critical for the development of targeted addiction therapies. Moreover, reward-related transcriptional gene regulatory mechanisms appear to be cell-type specific, with distinct roles for the NAc medium spiny neuron (MSN) subtypes. Therefore, to determine the causal contribution of cell-type specific, gene locus-targeted CREB recruitment in the differential pathogenesis of psychostimulant versus opiate abuse, I have engineered CRISPR technology as a vehicle for the conditional, locus-specific recruitment of CREB. I fused the nuclease- dead, RNA-guided, DNA-binding protein dCas9 to the active form of CREB (S133D), and I am able to direct CREB within targeted cell types to desired genomic regions to modulate transcription in vivo. Under the primary mentorship of Drs. Eric Nestler and Li Shen, I will receive training on in vivo, cell-targeted CRISPR delivery strategies, drug self-administration, and bioinformatic approaches. This training will allow me to use these highly specific manipulations to investigate a complete perspective on their differential contribution to psychostimulant versus opiate pathogenic volitional behaviors and transcriptional networks. I will validate and optimize my technology at the well-studied Fosb locus, allowing for meaningful interpretation of my data, and leverage the speed inherent to the CRISPR system to target a novel, bioinformatically predicted loci, Zfp189. In my independent phase, I plan to build upon my training to multiplex CREB binding to a novel drug self- administration identified gene network. This would establish the potential of using these physiologically- relevant manipulations to move beyond conventional monogenic manipulations in understanding complex neuropsychiatric syndromes. This training will aid in my transition into an independent investigator and yield novel insight on the differential causal pathogenic mechanisms of psychostimulant versus opiate abuse.